Project Summary/Abstract Musculoskeletal pain is a highly prevalent clinical problem, but males and females have been shown to experience marked differences in pain sensitivity/threshold, discrete expression patterns of pain related genes, differing susceptibility to chronic pain conditions, and dissonant post-injury analgesic responses. In both sexes, group III and IV muscle afferents serve as the chief input for muscle pain. However, these same afferents also are integral to the generation of the exercise pressor reflex (EPR), which stimulates exercise-induced increases in heart rate and blood pressure. Interestingly, males and females also show discrete response patterns in the EPR. Our preliminary data has shown distinct gene expression patterns between males and females in a preclinical model of ischemic myalgia, a widespread muscle pain state seen in disorders such as peripheral vascular disease and complex regional pain syndrome, among others. Specifically, we found a distinct pattern of dorsal root ganglion (DRG) gene expression between males and females for acid sensing ion channel 3 (ASIC3) and the proton-sensing heat transducer, TRPV1, after peripheral ischemia and reperfusion injury (I/R). Thus, we hypothesize that the group III and IV primary muscle afferents, serving as the peripheral regulator of both nociception and EPR, may be responsible for the complex interactions between pain, cardiovascular reflexes, and sex via differential upregulation of ASIC3 and TRPV1 in the DRGs. To study the molecular mechanisms underlying these sex differences, we will utilize our animal model of I/R injury which is known to dually generate pain-related responses in the affected limb and induce systemic EPR dysfunction, and couple it with an afferent-specific siRNA knockdown strategy of ASIC3 or TRPV1 in animals of both sexes. We will analyze muscle afferent function using a novel ex vivo forepaw muscle, median & ulnar nerves, DRG, and spinal cord recording preparation, which enables us to comprehensively phenotype these afferents in mice. Behavioral analyses of nociception and cardiovascular responses to exercise will also be performed in I/R or sham injured mice with or without siRNA knockdown. In Specific Aim 1, we will determine if upregulation of ASIC3 in the DRGs regulates I/R-induced alterations in muscle afferents leading to pain behaviors and altered EPRs in males but not females. In Specific Aim 2, we will perform similar assays but determine if upregulation of TRPV1 is responsible for observed molecular, electrophysiological and behavioral effects in females but not males. We predict that group III and IV primary muscle afferents mediate these processes in a sex-dependent manner, which may underlie the differential prevalence of pain conditions in men and women, and thus have implications for developing specific preventative care strategies and/or therapeutics.